Lynch A M, Meats J E, Best L, Tomlinson S
Department of Medicine, University of Manchester, U.K.
Biochim Biophys Acta. 1989 Jul 11;1012(2):166-70. doi: 10.1016/0167-4889(89)90091-8.
Intracellular pH (pHi) was measured in the insulin-secreting HIT-T15 cell line using the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). It was observed that the addition of a weak acid (e.g., acetate or propionate) caused a rapid decrease in pHi, followed by a slower recovery to the resting pH value. Conversely the addition of N4Cl caused an increase in pHi followed by recovery. The addition of amiloride caused a fall in pHi; however, in this case no recovery to basal pH levels was observed. Subsequent addition of a weak acid caused a further fall in pHi with no recovery. The addition of glucose caused a transient acidification followed by alkalinization. When glucose was added to cells which had been pretreated with amiloride, the initial acidification was not followed by recovery or alkalinization. Addition of glyceraldehyde, alpha-ketoisocaproate, lactate or pyruvate to HIT cells also resulted in intracellular acidification followed by recovery. Similarly, depolarisation of HIT cells by treatment with high K+ or with Ba2+ was associated with a pronounced fall in pHi, followed by a gradual recovery. Insulin secretion from HIT cells was stimulated by glucose, glyceraldehyde, alpha-ketoisocaproate, lactate, pyruvate and KCl, whilst amiloride and weak acids exerted only modest effects in the absence of glucose, but amiloride in particular markedly potentiated glucose-induced insulin release. Thus, HIT cells appear to have an amiloride-sensitive mechanism for the extrusion of protons, probably Na+-H+ exchange. Whilst intracellular acidification appears to potentiate secretory responses to nutrient stimuli, it seems unlikely that the activation of HIT cells by these nutrients occurs as a result of intracellular acidification. The mechanisms by which various nutrient and non-nutrient stimuli might exert distinct effects on pHi are discussed.
使用pH敏感荧光染料2',7'-双（羧乙基）-5'（6'）-羧基荧光素（BCECF）在胰岛素分泌型HIT-T15细胞系中测量细胞内pH值（pHi）。观察到添加弱酸（如乙酸盐或丙酸盐）会导致pHi迅速下降，随后缓慢恢复到静息pH值。相反，添加氯化铵会导致pHi升高，随后恢复。添加氨氯吡咪会导致pHi下降；然而，在这种情况下，未观察到恢复到基础pH水平。随后添加弱酸会导致pHi进一步下降且无恢复。添加葡萄糖会导致短暂酸化，随后碱化。当葡萄糖添加到用氨氯吡咪预处理过的细胞中时，最初的酸化之后没有恢复或碱化。向HIT细胞中添加甘油醛、α-酮异己酸、乳酸或丙酮酸也会导致细胞内酸化，随后恢复。同样，用高钾或钡离子处理使HIT细胞去极化与pHi显著下降相关，随后逐渐恢复。葡萄糖、甘油醛、α-酮异己酸、乳酸、丙酮酸和氯化钾刺激HIT细胞分泌胰岛素，而氨氯吡咪和弱酸在无葡萄糖时仅产生适度影响，但氨氯吡咪尤其显著增强了葡萄糖诱导的胰岛素释放。因此，HIT细胞似乎具有一种对氨氯吡咪敏感的质子外排机制，可能是钠-氢交换。虽然细胞内酸化似乎增强了对营养刺激的分泌反应，但这些营养物质激活HIT细胞似乎不太可能是细胞内酸化的结果。讨论了各种营养和非营养刺激可能对pHi产生不同影响的机制。
